The present invention relates to a method of removing pertussis endotoxin, a pertussis toxoid, and a method of producing the same.
Pertussis is a contagious infectious disease due to Bordetella pertussis and runs a serious course in infants and small children.
For the prevention of this disease, vaccines have heretofore been used. However, any vaccine prepared by using the whole organisms of B. pertussis produces intense adverse reactions such as fever and, to overcome this disadvantage, an acellular pertussis vaccine (ACP vaccine) substantially free of endotoxin (ET), which is mainly responsible for fever and other adverse reactions, by isolating the antiinfective fraction (hereinafter referred to sometimes as protective fraction) such as filamentous hemagglutinin (FHA), pertussis toxin (PT) and fimbriae has been developed and used.
The most crucial step in the production of an ACP vaccine is the separation of endotoxin (ET) from the antiinfective fraction and generally sucrose-gradient centrifugation has been utilized for this purpose (Japanese Unexamined Patent Publication No. 57-50925 which corresponds to EPC Publication No. 0047802).
Furthermore, as a technique for the removal of pyrogen, the method using calcium phosphate gel is known (Japanese Patent Publication No. 34-2149). It is also known that hydroxylapatite gel, a stabilized version of calcium phosphate gel, is effective in the separation of filamentous hemaggutinin (FHA) from pertussis toxin (PT) and that a substantially endotoxin free filamentous hemagglutinin can be separated from the pertussis toxin by chromatography on hydroxylapatite gel and further purification by affinity chromatography and sucrose-gradient centrifugation (Japanese Journal of Bacteriology, 38 (1), 423, 1983).
Sucrose-gradient centrifugation alone is capable of removing about 99.995% of the endoxin (ET) but as the crude antiinfective fraction-containing fluid is rich in endotoxin (ET), complete separation of the protective fraction from the endotoxin (ET) is hard to achieve and the yield of the antiinfective fraction is accordingly not high. Moreover, since the production volume is small and much cost and time are involved, the method is not satisfactory for commercial purposes.
On the other hand, mere treatment with calcium phosphate gel assures only a low endotoxin elimination rate of about 90 to 99.9% and is, therefore, not useful for practical purposes. It is possible, at the laboratory level, to separate and purify the filamentous hemagglutinin (FHA) fairly free of endotoxin in isolation from the pertussis toxin (PT) by a combination of hydroxylapatite column chromatography, affinity chromatography and sucrose-gradient centrifugation, but the method does not assure a commercially useful output. Furthermore, the technology involved is different in objective from the present invention which is directed to the removal of endotoxin (ET) from a fluid containing the protective fraction and endotoxin (ET).
Against the above technical background, we sought an efficient method for removing the endotoxin (ET) and found that a calcium phosphate gel treatment preceding the sucrose-gradient centrifugation results in a very neat separation of the protective fraction from endotoxin (ET) and, furthermore, affords improvements in both the volume of production and the rate of removal of endotoxin (ET) over sucrose-density centrifugation. These findings were followed by further research, which culminated in the completion of the present invention.